The complete and accurate replication of the genome is a central process in biology. This involves multiple processes as well as complex regulatory mechanisms that allow the temporally coordinated duplication of the chromosomes during each cell cycle. Pol ? plays a crucial role in DNA replication but in addition also is a major participant in DNA repair processes. A knowledge of the properties of human Pol ?, its regulation and the integration of its functions by protein-protein interactions with other components of the replisome and with DNA repair machinery is thus central to our understanding of human DNA replication and repair. Far less is known about human Pol ? than its more extensively studied counterpart in budding yeast, an important point because they differ in subunit composition. Our goals are directed toward dissecting the roles of the p12 and p68 subunits in Pol ? function. Our research is driven by the hypothesis that p12 directly affects the catalytic functions of the polymerase and exonuclease sites of Pol ?, while p68 plays a role as a scaffold for protein-protein interactions. In Aim 1 we take advantage of recent advances we have made in the expression of recombinant Pol ? and its subassemblies to make a rigorous biochemical comparisons of the core enzyme (p125/p50), core + p12, core + p68 and the holoenzyme. In Aim 2 we focus on determining the roles by identifying the sites of p12 that allow it to bind to the p125 and p50 subunits of the core enzyme, and the analysis of their individual effects We also will analyze the subunit interactions of Pol ? and PCNA. In Aim 3 we focus on the functions of p68, with the view that it acts a scaffold that mediates interactions of Pol ? with other proteins. We will investigate several hypotheses regarding the mechanisms by which phosphorylation-dephosphorylation regulates protein-protein interactions of p68, and also its role as a targeting protein that recruits protein phosphatase-1. Our studies could contribute to the understanding of cancer etiology, since it is more than likely that alterations in Pol ? could contribute to genomic instability. PUBLIC HEALTH RELEVANCE: Human Pol ? is one of the key enzymes that are involved in the duplication and repair of chromosomal DNA. Thus, its functions are highly important in replicating DNA without errors, as well as in repairing errors, so that mutations can be avoided. Our research is directed toward understanding how the different subunits of this enzyme contribute to the function of the catalytic core. These studies are crucial to understanding how it is able to perform DNA replication accurately, and how it interacts with other proteins that comprise the replication and repair machinery. Our work will also provide insights as to how Pol ? functions are regulated. There is currently a large gap in our knowledge of the human Pol ? enzyme, and our work is important to understanding how alterations in its functions could contribute to increased mutations in human cells, and to the etiology of cancer.